Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references (p. 109-119). / Eukaryotic cells are permeated by a three-dimensional network of entangled filamentous proteins termed the cytoskeleton. Like scaffolding, the cytoskeleton provides rigidity and resistance to deformation from forces transmitted to and from the cell membrane. In order to model the mechanics of the cytoskeleton, the interaction of individual structural proteins must be established. To this end the relationship between two critical proteins of the actin cytoskeleton is examined. Actin reversibly assembles into filaments that provide cells with shape and confer to the cell its mechanical properties. Filamin is an abundant actin-binding protein that efficiently cross-links actin filaments in large-angle orientations, requiring the lowest concentration to convert actin filaments into a cohesive gel. Filamin dimers are composed of two 24-repeat domains that come together like a V with an actin-binding region at each free end. Analysis reveals that the repeat domains of filamin are more flexible than the self-association region of the dimer. These findings dispute the initial claim that filamin is a rigid molecule. This thesis investigates the binding modality of filamin to actin. The structure of filamin bound to actin was compared to filamin in solution and immunogold molecules bound along the filamin rod were used to map the 3-D organization of filamin-actin junctions. There is evidence that filamin binds to actin at more sites than filamin's two established N-terminal actin-binding sites. These features, flexible repeat extensions, multiple-site binding, and a rigid self-association region, make filamin a potent cross-linking agent. / (cont.) The long flexible extensions allow filamin molecules to sample a large volume of cytoplasm in search of an actin target. The binding length of filamin along actin filaments provides a less-flexible linkage from actin to the rigid filamin self-association region, enabling reproducible large angles. At the same time, filamin brings actin filaments into close proximity, creating tight network entanglement, while filamin's angle prevents linked actin filaments from slipping into dense bundles as they do with short cross-linkers like [alpha]-actinin, instead maintaining a strong, disperse network. Tightly constrained junctions support recent entropic and enthalpic models of the cytoskeleton. / by Christopher A. Hartemink. / Ph.D.

Thesis (Ph.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 1999. / Includes bibliographical references. / by Jennifer Hartt Elisseeff. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 58-61). / Interviews were conducted to establish a baseline for how orphan drug forecasting is currently undertaken by financial market and industry analysts with the intention of understanding the variables typically accounted for in such a model. A literature search formed the basis of subsequent interviews conducted with experts from industry, payers, providers, legislators, patient groups, and the FDA. Discussion then focused on elements of the market which are poised to change in the short-term, how such changes might be reflected in existing models, and/or how these models may instead need to be modified to adapt to the new environment. We hypothesized that impending changes in the healthcare sector would indeed impact the legitimacy of current forecasting models, and that significant changes would need to be introduced to account for these new market forces. Our hypothesis, however, was not confirmed, in that although much of the literature and, indeed, public outcry over rising healthcare costs in general and drug prices in particular make a strong case for implementing changes in the orphan market via payers, government, or other actors, an assessment of healthcare experts regarding market changes over the next five years revealed a general consensus that meaningful change will likely not occur during this timeframe for orphan products, with the exception of a possible increase in pharmacoeconomic requirements for drugs which are only marginally effective. Thus, current orphan drug forecasting models constructed for use by financial and industry analysts correctly avoid discounting for these potential changes, as they will likely not face significant changes in the US until closer to a ten year time horizon. Potential exceptions to this conclusion depend on implementation and regulatory treatment of the fields of personalized medicine and gene therapy, as developments in these areas may closely interact with existing orphan drug legislation. Our results have significant implications for all companies and stakeholders entering or currently operating in the orphan market, and open the door for further quantitative and qualitative analysis. / by Joshua Gottlieb. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references. / Many brain tumors are localized deeply and are currently surgically inaccessible without causing severe damage to the overlying structures of the brain. The current spectrum of non-invasive methods for treating such tumors includes radiotherapy, which requires exposure to ionizing radiation, and chemotherapy, which is systemically toxic. However, these tumors may also potentially be attacked by focusing highly intense ultrasound onto them. Focused ultrasound surgery is without the side effects of radiotherapy and chemotherapy, and the therapeutic effect of ultrasound therapy can be monitored in real- time using the proton chemical shift MRI technique. However, in order for brain tumors to be treated non-invasively, the ultrasound must be focused onto the targeted brain tissue through the intact cranium. Transcranial focusing of ultrasound is a longstanding and difficult problem as skull is a highly heterogeneous material. As the ultrasound field propagates through the bones of the skull, it undergoes substantatial distortion due to the variations in density and speed of sound therein. There is substantial individual variation in skull size, thickness and composition. Furthermore, the acoustic attenuation coefficient in bone is high, so the skull may also be heated by the ultrasound propagating through it. This thesis contains novel simulation techniques for analyzing transcranial acoustic propagation and for analyzing the temperature changes so produced in the brain, skull and scalp. These techniques have also been applied to modeling non-invasive treatment of the liver, and to producing therapeutic ultrasound fields that harness non-linear acoustic effects advantageously. / (cont.) The thesis also contains unified models for the speed of sound and the acoustic attenuation coeffiecient in human skull. These models were generated by combining genetic optimization algorithms, acoustic propagation modeling and empirical measurement of intracranial ultrasound fields; they are valid across the full range of trabecular and cortical cranial bone. / by Christopher W. Connor. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references (leaves 63-64). / The United States population is aging, and the need for novel approaches to treat and manage disease continues to grow. Among the diseases that will impact this population, cancer remains a therapeutic area with significant unmet need. Pharmaceutical and biotechnology industries must continue to meet revenue and income growth expectations and will become increasingly dependent on novel drugs in their pipelines. In order for the pharmaceutical and biotechnology industries to meet the demands of both patients and shareholders, productivity in the research and development process will need to improve significantly. In order to understand how best to improve the drug discovery and development process, it is important to identify potential sources of innovation throughout the process. Among these, an important consideration is to understand the paths that molecules take through the discovery and development process. / (cont.) This thesis used the marketplace of oncology drugs in development to test the hypothesis that novel molecules largely originate in academia, are developed by biotechnology companies and ultimately are licensed to pharmaceutical companies for commercialization. This thesis analyzed a database of 364 unique oncology small molecules and biologics entering Phase I clinical development between 1991 and 2002. / by Jorge Cesar Conde. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Includes bibliographical references (p. 107-121). / The clinical application of chemotherapy to brain malignancies has been severely limited because many potential therapeutic agents are typically unable to penetrate the blood-brain barrier (BBB). A novel approach to overcome this barrier uses focused ultrasound to induce localized BBB disruption in a targeted region of the brain and magnetic resonance imaging (MRI) to guide and monitor the procedure. The purpose of this thesis was to develop a technique using MRI-guided focused ultrasound for trans bbb drug delivery applications. This thesis demonstrates that MRI-guided focused ultrasound can be used to achieve consistent and reproducible BBB disruption without invasive craniotomy in rats, to enable doxorubicin to accumulate in normal brain at clinically therapeutic levels, and to increase the antitumoral efficacy of doxorubicin in a rodent model of aggressive glioma. Using a microbubble-based ultrasonographic contrast agent, focal BBB opening was consistently achieved using transcranial focal pressures of 1.2 MPa or greater; locations in the posterior brain exhibited consistent BBB disruption with applied focal pressures of 0.8 MPa or greater. When combined with systemic administration of liposomal doxorubicin, we achieved local drug concentrations of 900 ± 300 ng/g tissue in the brain with minimal tissue effects, and up to 5400 ± 700 ng/g tissue with more significant tissue damage, while accumulation in non-targeted contralateral brain tissue remained significantly lower (p < 0.001). In addition, MRI signal enhancement in the sonicated region correlated strongly with doxorubicin concentration in tissue (r = 0.87), suggesting that contrast-enhanced MRI may provide useful feedback on drug penetration. / (cont.) Finally, glioma-bearing rats treated with ultrasound-enhanced chemotherapy exhibited significantly longer median survival times (31 versus 25 days; p = 0.0007) and slower tumor growth (average tumor volume doubling time, 3.7 ± 0.5 days, versus 2.3 ± 0.3 days) than nontreated rats; rats which received standard intravenous chemotherapy showed no significant difference in survival or tumor growth rate. In sum, this thesis research provides pre-clinical data toward the development of MRI-guided focused ultrasound as a noninvasive method for the delivery of agents such as doxorubicin across the BBB to treat patients with diseases of the central nervous system. / by Lisa Hsu Treat. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2003. / Includes bibliographical references (p. 40-44). / There is a clear and present need to improve the quality of diabetes care. Information technology can be used as a means to that end. In this article, we discuss the design and implementation of a web-based diabetes application. We show the role of modeling clinical workflow in the design philosophy of our application, and summarize our application's features and usage. Next, we describe observations made during and after design and implementation, and how they relate to the informatics literature. Finally, we elaborate on the paradigm of feedback control systems, its parallels with the design philosophy of our application, and its use as an organizational framework for the roles of information technology in diabetes care. / by Anil Kumar Dubey. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references (leaves 81-86). / Bacterial infections represent a significant source of morbidity and mortality. Biofilms and antibiotic resistance pose challenges to our future ability to treat bacterial diseases with antibiotics (1). Bacteria frequently live in biofilms, which are surface-associated communities encased in a hydrated extracellular polymeric substances (EPS) matrix (2, 3). Biofilms are crucial in the pathogenesis of many clinically-important infections and are difficult to eradicate because they exhibit resistance to antimicrobial agents and removal by host immune systems (4). Antibiotics can even induce biofilm formation (5, 6). The development of antibiotic-resistant bacteria is also a growing medical problem. Antibiotic resistance genes can be acquired by horizontal gene transfer and passed vertically to later generations (7). Antibiotic resistance can also result from persistence, a phenomena in which a subpopulation of cells can withstand antibiotic treatment without containing antibiotic-resistance genes (8). These problems, coupled with decreasing output of new antibiotics, have highlighted the need for new treatments for bacterial infections (1, 9-12). I developed three novel strategies for attacking bacterial biofilms and antibiotic resistance using synthetic biology. To remove biofilms, I engineered bacteriophage to express a biofilm degrading enzyme during infection to simultaneously attack biofilm cells and the biofilm EPS matrix. These enzymatically-active bacteriophage substantially reduced biofilm cell counts by 4.5 orders of magnitude (-99.997% removal), which was about two orders of magnitude better than that of non-enzymatic phage. To address antibiotic-resistant bacteria, I targeted gene networks with synthetic bacteriophage to create antibiotic adjuvants. / (cont.) Suppressing the SOS network with engineered bacteriophage enhanced killing by ofloxacin, a quinolone drug, by over 2.7 and 4.5 orders of magnitude compared with control bacteriophage plus ofloxacin and ofloxacin alone, respectively. I also built phage that targeted multiple gene networks and demonstrated their effectiveness as antibiotic adjuvants. Engineered bacteriophage reduced the number of antibiotic-resistant bacteria and performed as strong adjuvants for other bactericidal antibiotics such as aminoglycosides and P-lactams. Finally, I designed synthetic in vivo sensors for antibiotic-resistance genes that can be coupled with effector components to kill cells carrying resistance genes or to block horizontal transmission of those genes. My work demonstrates the feasibility and benefits of using engineered bacteriophage and synthetic biology constructs to address the dual threats of bacterial biofilms and antibiotic-resistant bacteria. / by Timothy Kuan-Ta Lu. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, June 2005. / Includes bibliographical references. / Chapter 1. An Introduction to Self-Assembled Monolayers & Surface Characterization A brief summary of the formation, structure, and characterization techniques of self assembled monolayers (SAMs) is described. The characterization techniques include contact angle goniometry, ellipsometry, grazing-angle Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), sum-frequency generation spectroscopy (SFG), and atomic force microscopy (AFM). Chapter 2. A Reversibly Switching Surface The design of surfaces that exhibit dynamic changes in interfacial properties such as wettability in response to an electrical potential is described. The change in wetting behavior was caused by surface-confined, single-layered molecules undergoing conformational transitions between a hydrophilic and a moderately hydrophobic state. Reversible conformational transitions were confirmed both at a molecular level using sum-frequency generation spectroscopy and at a macroscopic level using contact angle measurements. This type of surface design enables amplification of conformational transitions at a molecular level to macroscopic changes in surface properties without altering the chemical identity of the surface. Such reversibly switching surfaces may open new opportunities in interfacial engineering.Chapter 3. A Synthetic Chemical Route for the Formation of Homogeneously- Mixed Self-Assembled Monolayers A novel way to produce self-assembled monolayers (SAMs) uniformly mixed on the molecular length scale is described. / (cont.) Initially, a precursor SAM was formed from molecules that are derived from 16-mercaptohexadecanoic acid (MHA) and contain a globular end group. Self-assembly of these molecules resulted in a SAM that is densely packed with respect to the space-filling end groups, but shows low-density packing with respect to the hydrophobic chains. Subsequent cleavage of the space-filling end groups established a low-density SAM of MHA. A mixed monolayer of MHA and n-butanethiol was formed by backfilling the low-density monolayer of MHA with the corresponding alkanethiol. The new "mixed" SAM was characterized by optical ellipsometry, contact angle goniometry, X-ray photoelectron spectroscopy (XPS), Fourier Transform Infrared Spectroscopy (FT-IR), cyclic voltammetry (CV), and reductive desorption voltammetry. The results indicate a uniformly mixed monolayer as compared to a SAM generated by coadsorption of mixtures of the same MHA and n-butanethiol molecules. This approach provides a way to produce SAMs that are uniformly mixed using a synthetic chemical route, which affords considerable flexibility in composition and also in the ratio of the different molecules in the mixed SAM. Chapter 4. / (cont.) Design of Oligonucleotide Arrays Using Homogeneously Mixed Self - Assembled Monolayers We have employed two quantitative techniques, quart-crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance imaging (SPR) to quantify the hybridization efficiency of a 25-mer oligonucleotide probe to two different surfaces: a dense 16-mercaptohexadecanoic acid self-assembled monolayer (MHA SAM) and a homogeneously-mixed (HM) SAM generated from the method described in Chapter 3 that allows for regular spacing of functional -COOH groups. This reduced density of functional groups led to reduced attachment of oligonucleotide probes to the surface, increasing the area per probe, and allowed more space in which complimentary sequence can bind. Reducing the density of immobilized probes led to the improvement in hybridization efficiency as demonstrated in both SPR and QCM-D results, which are comparable to previous reports. Our method paves the way for customizing binding efficiency and target probe density based on the distance between functional groups. By changing the headgroup size of the precursor monolayer, different distances between functional group can be formed, allowing for an ability to tailor distances between molecules. This method may allow for improvement in DNA array technology.Chapter 5. Long-Term Stability of Self-Assembled Monolayers in Biological Media The study of long term stability of self-assembled monolayer (SAM) in biological media is of importance in evaluating its usefulness for applications in implantable biochips, biosensors, or biological microelectromechanical system (bioMEMs) devices for drug delivery. / (cont.) To minimize biofouling effects, researchers have investigated protein/cell adhesion resistant surface-bound materials such as poly(ethylene glycol) or oligo(ethylene glycol) terminated self-assembled monolayers. However, no long term study in biological media has been done. To address the issue of moderate to long-term stability of SAMs for bioMEMS device modification, alkanethiol and oligo(EG) terminated alkanethiol monolayers were prepared and studied after immersion in either phosphate buffer saline (PBS) or calf serum. Here, undecanethiol (CllH23SH) and tri(ethylene glycol) terminated undecanethiol (HO(C2H40)3C H22SH) self-assembled monolayers (SAMs) on clean gold surfaces were prepared and characterized. The SAMs were then immersed into either phosphate buffered saline (PBS) or calf serum. The SAM samples were emmersed and investigated using several analytical techniques at numerous points over the next 35 days. Contact angles and current densities in voltammetry changed dramatically for the PBS samples over the time period, particularly after 21 days. Results indicate substantial loss of the integrity of the SAM. Similar alterations with time were observed for the calf serum samples in both contact angle and voltammetry measurements. X-ray photoelectron spectroscopy indicates that the likely origin is desorption of the alkanethiol moiety as evidenced by appreciable loss of the S 2p signal after 35 days. Additionally, this work may serve as a starting point for further studies of surface chemical modification methods for moderate to long-term minimization of biofouling for in vivo applications. / by Thanh-Nga T. Tran. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references. / Superior semicircular canal dehiscence (SCD) syndrome is a recently defined clinical disorder in which patients present to the clinic with vestibular symptoms, auditory symptoms, or both. Understanding the effect of SCD, a hole in the bony superior canal, on hearing will broaden our understanding of the mechanics of the inner ear and lead to better diagnosis and treatment of SCD syndrome. We evaluate the effect of SCD on cochlear responses to both air- and bone- conducted sound. In chinchilla SCD produces reversible changes in cochlear potential: an increased sensitivity to bone-conducted sound and a decreased sensitivity to air-conducted sound. Such differences in air- and bone-conducted sound (air-bone gaps) are typically associated with a conductive hearing loss due to middle-ear pathology; however, a SCD is an inner-ear pathology. We hypothesize that the SCD acts as a 'third window' into the inner ear, shunting volume velocity away from the cochlea and through the dehiscent canal, altering cochlear responses to sound. To qualitatively evaluate this hypothesis we measured sound-induced fluid motion within the SCD as well as the effect of SCD on sound-induced stapes velocity and middle-ear input admittance. / (cont.) Our results are consistent with the hypothesis that the SCD introduces a low-impedance shunt pathway. To quantitatively evaluate the third-window hypothesis we developed an anatomically and physiologically constrained lumped-element mechano-acoustic model that predicts the effect of SCD on cochlear responses. Our model also predicts the effect of anatomical variations, such as dehiscence size and location, on auditory sensitivity. This work demonstrates that an air-bone gap can be caused by pathological changes in inner-ear mechanics. Additionally, our model provides a framework that will be of direct clinical benefit in understanding the variable effects of SCD among patients. / by Jocelyn E. Songer. / Ph.D.